Off-target reactivity is the single largest cause of failed preclinical drug programs, accounting for 62% of all failures. Despite their presumed specificity, emerging data suggest that up to 20% of lead MAbs display off-target binding, often against completely unpredicted and unrelated targets. Comprehensive specificity profiling is needed to better predict off-target binding of MAbs and de-risk preclinical candidates prior to IND submission.
Integral Molecular’s The Membrane Proteome Array (MPA) was developed to provide detailed MAb specificity analysis against native-conformation epitopes from a library of > 4,500 unique human membrane proteins. We used the MPA to screen a panel of novel MAbs raised against SLC2A4 (GLUT4), a 12-TM insulin-sensitive glucose transporter. MPA analysis and subsequent validation demonstrated no SLC2A4 MAb cross-reactivity against related transporters. However, one of the MAbs from the panel screened on the MPA, MAb LM052, interestingly showed low-level reactivity to the unrelated signaling protein Notch1. This was completely unexpected, as SLC2A4 and Notch1 are structurally unrelated (12-TM versus 1-TM) and share less than 6% sequence identity. Rakocevic et al., 2021, bioRxiv)
High-resolution epitope mapping using our premier Shotgun Mutagenesis platform gave insight into the cross-reactivity of LM052 for Notch1. The epitope for LM052 was mapped at amino acid resolution to a loop-constrained LGXXGP sequence on SLC2A4. Despite being unrelated in sequence and structure to SLC2A4, the exact same LGXXGP motif was discovered to be shared on a disulfide-constrained region of Notch1, giving was to LM052’s cross-reactivity detected by the MPA.
Importance of Specificity Testing
The unpredicted cross-reactivity of LM052 with Notch1, a completely unrelated protein to the SLC2A4 target protein, attests to the need for comprehensive MAb target profiling and the limitations of specificity testing against family members alone. Our findings indicate that polyspecificity should be assessed prior to the start of preclinical development and selection of lead candidates to avoid downstream clinical failures. (Tucker et al, 2018, PNAS) , bioRxiv)